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HPFM3 Panorama™ Human Kinase V1 Array Panorama™ Human Kinase v1 Array Technical Bulletin Catalog Number HPFM3 Panorama™ Human Kinase v1 Array Table of Contents Introduction ......................................................................................................................................... 1 Panorama Array Technology ................................................................................................................. 1 Kit Contents ........................................................................................................................................ 1 Storage Conditions .............................................................................................................................. 2 General Recommendations .................................................................................................................. 2 Kit Components ................................................................................................................................... 3 Protein Arrays .................................................................................................................................................. 3 Anti-phosphotyrosine-Cy5................................................................................................................................ 4 Anti-c-Myc-Cy3 ................................................................................................................................................ 4 Control Assays ..................................................................................................................................... 4 Protocol for Autophosphorylation Assay .......................................................................................................... 4 Protocol for the Anti-c-Myc-Cy3 Binding Assay ................................................................................................. 6 Scanning .......................................................................................................................................................... 7 Data Analysis .................................................................................................................................................. 7 Data Normalization .......................................................................................................................................... 8 Standard Normalization.................................................................................................................................... 8 Guidelines for Different Assay Types ..................................................................................................... 9 General Recommendations .............................................................................................................................. 9 Phosphorylation on Arrays Using Exogenous Kinases ........................................................................................ 9 Assays Utilizing Radioactive ATP ....................................................................................................................... 9 DNA Binding Assays ....................................................................................................................................... 10 Antibody Binding Assays ................................................................................................................................ 10 Protein:Protein Interactions ............................................................................................................................ 10 Post-translational Modifi cation ....................................................................................................................... 10 Troubleshooting Guide ....................................................................................................................... 11 Appendices ........................................................................................................................................ 12 Appendix A: Protein Array Orientation ........................................................................................................... 12 Appendix B: Panorama Human Protein Function Array Kinase v1—Protein Identities ....................................... 13 Control and Marker Proteins .......................................................................................................................... 17 Dilution Series ................................................................................................................................................ 17 References ......................................................................................................................................... 19 Relevant Patents ................................................................................................................................ 19 Introduction The Panorama™ Human Kinase v1 Array contains 152 kinase proteins and specifi c controls. Kinases are categorized into familes based on their structure and activity and are known to regulate the majority of cellular pathways, especially those involved in signal transduction and transmission of signals within the cell. Loss of kinase function has been linked to many human diseases, such as cancer1, and is known to affect cascades of reversible phosphorylation. Kinases are attractive targets for therapeutic drugs because they have similar structures and highly conserved ATP binding sites. Drugs which inhibit specifi c kinase activity such as Gleevec® and Iressa® are currently in clinical use for the treatment of numerous malignancies. Panorama Array Technology The kinases on the Panorama Human Kinase v1 Array were expressed in Sf9 insect cells and affi nity purifi ed directly on the array via their biotin tag. As a result of the proprietary BCCP (biotin-carboxyl carrier protein) tagging technology, all kinases are presented in a similar orientation while providing a 50 Å spacer arm to maximize the opportunity for sites to interact with binding partners2,3,4. Open reading frames (ORFs) are cloned in frame with two tag sequences at the C terminus encoding the BCCP tag and the c-Myc epitope (EQKLISEEDL), which can be used to visualize and quantitate the proteins on the array. To ensure fi delity, clones are sequence-verifi ed immediately prior to expression in Sf9 insect cells. During expression, the BCCP tag is biotinylated only when it is correctly folded5. All expressed proteins are assayed for incorporation of biotin, and Western blot analysis is used to determine molecular weight, confi rm biotinylation, and establish full-length protein has been expressed. Biotinylation of BCCP occurs at a single surface-exposed lysine residue approximately 50 Å from the attachment to the fusion protein. The BCCP-biotin-fusion proteins are captured on the array surface via a streptavidin-biotin interaction with BCCP acting as a spacer between the array substrate and the fusion protein. BCCP-biotin provides a single-point high-affi nity anchor so that all proteins on the array are in the same orientation. As a result, the arrayed proteins are not sterically or functionally hindered by multiple non-specifi c interactions with the surface and are freely available to interact with biochemical probes presented in solution, thereby minimizing non-specifi c interactions6. Panorama functional protein arrays are fabricated on borosilicate glass slides that display high chemical resistance, low auto-fl uorescence, and excellent surface uniformity. The slides are cut by a laser to minimize particle contamination. The slides are then coated with streptavidin that is covalently attached to a permeable three-dimensional coating comprised of a cross-linked matrix with low non-specifi c protein-binding. The format is compatible with conventional microarray scanners and instrumentation. Kit Contents Product Cat. No. Size Panorama Human Kinase v1 Microarrays P2374 2 each Anti-phosphotyrosine-Cy5 T3576 10 µL Anti-c-Myc-Cy3 C6594 10 µL Functional Assay Buffer C0492 150 mL Assay Buffer A1105 150 mL Bovine Serum Albumin (BSA) A3059 2 3 200 mg 1M Dithiothreitol (DTT) 646563 1 ampule 1 Product Cat. No. Size 100 mM ATP A6559 100 µL quadriPERM® culture vessel Z376760 2 each HybriSlip™ H0784 10 each Pap Jar P8123 4 each 50 mL Conical Centrifuge Tubes C8296 2 each Panorama Kinase Array v1 Analysis Workbook and K4139 1 each GAL fi le (CD) Materials Required But Not Provided • High-purity water • Powder-free gloves • Microarray scanner or fl uorescence imager • Microarray analysis software • Forceps (fi ne and blunt-ended) • Centrifuge • Shaking incubator • Orbital shaker • Lint free tissue paper (e.g., Kimwipes) Storage Conditions Proteins on the array are sensitive to heat and oxidation. To preserve protein activity, the arrays are shipped on dry ice in screw-capped Pap jars and fi lled with 30 mL of storage buffer containing dithiothreitol and glycerol. Upon receipt, store the kit at –20 °C until use. The storage buffer for the protein arrays may be frozen upon arrival due to the dry ice used for shipping, but will thaw gently when placed at –20 °C. Once the storage buffer has thawed, open Pap jars only prior to use. General Recommendations a. The array area covers most of the slide surface; therefore, extreme care is needed in handling the arrays. Remove the arrays from their storage buffer by the labeled end using blunt-ended forceps. Do not touch the unprotected portion of the
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